In eukaryotic cells, mitosis is initiated following the activation of a protein kinase known as MPF, the M-phase specific histone kinase or more simply as the M-phase kinase. This kinase consists of at least three subunits; the catalytic subunit (cdc2), a regulatory subunit (cyclin B) and a low molecular weight subunit (p13-Sucl).
There is much interest in the regulation of the phosphatase which dephosphorylates cdc2 because of its role in the activation of MPF. Genetic studies in fission yeast have established that the cdc25 gene function is essential for the initiation of mitosis, Nurse, P. et al., Mol. Gen. Genet. 146:167-178 (1976). The cdc25 gene product serves as a rate-determining activator of the cdc2 protein kinase, Russell, P. and P. Nurse, Cell 45:145-153 (1986); Ducommun, B. et al., Biochem. Biophys. Res. Common. 167:301-309 (1990); Moreno, S. et al., Nature 344:549-552 (1990)). Mutant cdc2-F15, whose product cannot be phosphorylated on tyrosine, bypasses the requirement for cdc25 protein function, Gould, K. and P. Nurse, Nature 342:39-45 (1989)). Additional work suggested that cdc25 is a cdc2 phosphatase, Kumagai, A. and W. G. Dunphy, Cell 64:903-914 (1991) and Strausfeld, U. et al., Nature 351:242-245 (1991).
Apparently cdc25 acts as a cdc2 phosphatase which dephosphorylates tyrosine and possibly threonine residues on p.sub.34 cdc2 thus regulating MPF activation, Dunphy, W. G. and A. Kumagai, Cell 67:189-196 (1991) and Gautier, J. et al., Cell 67:197-211 (1991). Because cdc25 phosphatases are responsible for the dephosphorylation and activation of cyclin-dependent protein kinases, they help control cell cycle progression.
As a cell cycle specific phosphatase, cdc25B is believed to be crucial for progression from G2 through mitosis. To study this protein and/or develop a screen using this protein one requires the isolation of the active catalytic domain of cdc25B. Unfortunately, the native full length cdc25B is difficult to obtain due to its sensitivity to proteolysis and its low abundance in mammalian cells On the other hand, a GST fusion protein with cdc25B has been described which has activity. The latter protein has partial solubility and measurable activity as a phosphatase, but as a fusion protein is not amenable to structural analyses including crystallographic studies. To date, successful removal of the GST moiety from a full length fusion protein of cdc25B, with subsequent isolation of full length cdc25B without a GST tag, has not been reported. There have been attempts to make smaller recombinant catalytic domains of cdc25.B, including one recently described by Horiguchi et al. The latter recombinant form includes a GST fusion partner and codes for amino acids 355-566, Takashi Horiguchi, et al., Biochemical Pharmacology, Vol. 48 pp. 2139-2141, (1994), incorporated by reference, but this construct appears to result in low protein yields and the protein product is poorly soluble with low activity. The observation of low activity of recombinant forms of cdc25B expressed in E. coli is most likely attributable to improper enzyme folding.
Stable recombinant forms of cdc25B are needed that have improved activity making them suitable for use in enzyme assays with improved solubility characteristics. Stable recombinant forms of the protein that are capable of easy manipulation for crystallography studies in order to better understand and characterize these types of phosphatases by structural analyses and models are also needed. This invention provides macro molecules having these and other desirable characteristics.